Use of a high-temperature inducible promoter in potato breeding regulation

Abstract

This invention relates to the fields of plant genetic engineering and potato breeding technology, and particularly to the application of a heat-inducible promoter in potato breeding regulation. This invention provides transgenic lines expressing SP6A and SP6AM induced by pHSP101 and pHSP70. Compared with the wild type, these transgenic lines exhibit improved tuber formation ability at both normal and high temperatures. This invention evaluated the tuber formation phenotype of the induced transgenic lines using two methods (in vitro high-sugar tuber induction system and conventional soil-cultured potatoes). The results showed that the transgenic lines had tuber formation ability under high temperatures compared to the wild type. This invention used qRT-PCR and laser confocal microscopy to detect the transcriptional and protein expression levels of the SP6A gene in the heat-induced lines, demonstrating that the promoters pHSP70 and pHSP101 respond to high temperatures to promote potato tuber formation, providing genetic materials and a theoretical basis for elucidating the mechanism of heat-resistant tuber formation in potatoes and for heat-resistant improvement breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and potato breeding technology, and in particular to the application of a high-temperature inducible promoter in potato breeding regulation. Background Technology

[0002] Potatoes are the fourth largest food crop after wheat, rice, and corn. Compared to other grains, potatoes are lower in calories and rich in dietary fiber, which can lower serum cholesterol and prevent gallstones, obesity, and various chronic diseases caused by obesity. Potatoes are a cold-loving plant; in high-temperature environments, their reproductive growth and tuber formation are severely affected. Nighttime temperatures of 25°C and above inhibit tuber formation, and stem and leaf growth ceases when the ambient temperature reaches 39°C. Therefore, studying the molecular mechanisms of potato response to high temperatures and further improving potato heat resistance through molecular biology and genetic engineering techniques has significant theoretical and practical value for breeding new heat-resistant and high-yielding potato varieties.

[0003] Heat shock proteins are strongly induced and rapidly accumulate under high temperatures, playing a crucial role in plant cell responses to heat stress. Based on their molecular weight, heat shock proteins can be classified into HSP100, HSP90, HSP70, HSP60, HSP40, and sHSP. Under heat stress, HSPs act as molecular chaperones in protein folding, transport, and degradation. The function of HSPs in potato heat tolerance has also been reported. HSP20s are a very important class of heat stress response genes. Currently, 48 HSP20s members have been identified in potatoes, and most of them can be rapidly and extensively expressed under heat stress.

[0004] SP6A is an important inducing factor for potato tuber formation. A miRNA (SUPPRESSING EXPRESSION OF SP6A, SES) targeting the transcription product of StSP6A exists in potatoes, and studies have shown that SES may be involved in the temperature-dependent regulation of StSP6A expression. Previous studies on high-temperature treatment of StSP6A overexpression lines found that overexpression of StSP6A with the 35S promoter restored tuber formation ability under high temperatures, but did not restore tuber yield. Researchers speculate that this may be related to the inhibition of sugar transport by high temperatures. Although the 35S promoter from cauliflower mosaic virus can widely initiate the expression of downstream genes with high abundance, this widespread and persistent gene expression can also affect plant growth and development to some extent. Numerous studies have shown that many overexpression lines constructed using the 35S promoter exhibit growth-inhibited phenotypes, and the 35S promoter is not active in all tissues and cells. Finally, studies have shown that the 35S promoter can not only regulate the expression of its driver genes but also affect the expression of neighboring genes, which further limits the use of the 35S promoter.

[0005] Therefore, exploring the heat resistance mechanism of potatoes and creating new potato materials that induce tuber formation at high temperatures remains crucial. Summary of the Invention

[0006] To address the aforementioned problems, the purpose of this invention is to provide an application of a high-temperature inducible promoter in potato breeding regulation.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The first objective of this invention is to provide an application of a high-temperature inducible promoter in potato breeding regulation, wherein the high-temperature inducible promoter is selected from pHSP101 or pHSP70.

[0009] The nucleotide sequence of pHSP101 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of pHSP70 is shown in SEQ ID NO.2.

[0011] In one embodiment of the present invention, the high-temperature inducible promoter is used to promote tuber formation in potatoes at both room temperature and high temperature and / or to increase the starch content of potato tubers.

[0012] The second objective of this invention is to provide an application of a heat-inducible expression vector in potato breeding regulation, wherein the heat-inducible expression vector comprises the above-mentioned high-temperature inducible promoter and potato tuber gene;

[0013] The potato tuber gene is selected from either the SP6A gene or the SP6AM gene.

[0014] The nucleotide sequence of the SP6A gene is shown in SEQ ID NO.3;

[0015] The nucleotide sequence of the SP6AM gene is shown in SEQ ID NO.5.

[0016] In one embodiment of the present invention, the heat-inducible expression vector is used to promote tuber formation in potatoes at room temperature and high temperature and / or increase the starch content of potato tubers.

[0017] A third objective of this invention is to provide an application of heat-induced host cells in potato breeding regulation, wherein the heat-induced host cells include the aforementioned heat-induced expression vector.

[0018] In one embodiment of the present invention, the heat-induced host cells are used to promote tuber formation in potatoes at room temperature and high temperature and / or increase the starch content of potato tubers.

[0019] The fourth objective of this invention is to provide a method for preparing a potato strain with high-temperature tuber formation ability, comprising the following steps:

[0020] (S1) The high-temperature inducible promoter and the potato tuber gene were inserted into the basic vector to construct a recombinant vector containing the high-temperature inducible promoter and the potato tuber gene;

[0021] (S2) A recombinant vector containing a high-temperature inducible promoter and a potato tuber gene was transferred into Agrobacterium and then infected with potato micro-tuber chips to induce bud differentiation, thus obtaining a potato line with the ability to form tubers at high temperatures.

[0022] The high-temperature inducible promoter is selected from either pHSP101 or pHSP70.

[0023] The nucleotide sequence of pHSP101 is shown in SEQ ID NO.1;

[0024] The nucleotide sequence of pHSP70 is shown in SEQ ID NO.2.

[0025] In one embodiment of the present invention, in step (S1), the potato tuber gene is selected from either the SP6A gene or the SP6AM gene;

[0026] The nucleotide sequence of the SP6A gene is shown in SEQ ID NO.3;

[0027] The nucleotide sequence of the SP6AM gene is shown in SEQ ID NO.5.

[0028] In one embodiment of the present invention, in step (S1), the base carrier is an EZR carrier;

[0029] In step (S2), the Agrobacterium is Agrobacterium GV3101.

[0030] The fifth objective of this invention is to provide a potato strain capable of tuber formation at high temperatures, prepared by the above method, which can form tubers normally at 28–31°C.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention provides transgenic lines expressing SP6A and SP6AM induced by pHSP101 and pHSP70, which can form tubers normally under high temperatures. The tuber-forming phenotype of the induced transgenic lines was evaluated using two methods (in vitro high-glucose tuber induction system and conventional soil-cultured potatoes). The results showed that the transgenic lines have the ability to form tubers under high temperatures compared to the wild type. This invention used qRT-PCR and laser confocal microscopy to detect the transcriptional and protein expression levels of the SP6A gene in the heat-induced lines, demonstrating that the promoters pHSP70 and pHSP101 respond to high temperatures and promote potato tuber formation, providing genetic materials and a theoretical basis for elucidating the potato heat tolerance mechanism and for heat tolerance improvement breeding. Attached Figure Description

[0033] Figure 1 This example illustrates the high-temperature expression of potato HSPs genes and the analysis of promoter sequence characteristics.

[0034] Figure 2 The sequence characteristics of the heat-induced expression vector constructed in the examples;

[0035] Figure 3 The laser confocal microscopy method was used in the examples to identify the expression of SP6A-GFP / SP6AM-GFP fluorescent proteins in the heat-induced expression lines.

[0036] Figure 4 This is the in vitro tuber formation phenotype of the heat-induced expression lines in the examples after high-temperature treatment.

[0037] Figure 5 This example demonstrates the identification of tuber formation phenotype and gene expression analysis of soil-grown seedlings of heat-induced expression lines.

[0038] Figure 6 This example illustrates the identification of tuber formation phenotype in field trials conducted under high-temperature weather for heat-induced expression lines.

[0039] Figure 7 This example illustrates gene expression analysis of heat-induced expression lines used in field experiments. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0041] In the following embodiments, the potato E3 is E'Potato No. 3. Unless otherwise specified, all reagents used are commercially available, and all detection methods and techniques used are conventional detection methods and techniques in the field.

[0042] In the following examples, the nucleotide sequence of promoter pHSP101 is shown in SEQ ID NO.1; the nucleotide sequence of promoter pHSP70 is shown in SEQ ID NO.2; the nucleotide sequence of SP6A is shown in SEQ ID NO.3, and the protein sequence is shown in SEQ ID NO.4; the nucleotide sequence of SP6AM is shown in SEQ ID NO.5, and the protein sequence is the same as that of SP6A.

[0043] In the following examples, the pHSP101:SP6A-1 strain represents a heat-induced strain containing the expression vector pHSP101:SP6A and numbered 1, which can be abbreviated as "101A-1"; other similar representations can refer to the above definition.

[0044] Example 1

[0045] This embodiment provides analysis of the high-temperature expression and sequence characteristics of potato HSPs.

[0046] Tissue culture seedlings E3 that had grown for 3 weeks were subjected to high-temperature treatment under the following conditions: 30℃ light / 28℃ darkness, with a light intensity of 83 μmol / m². –2 s –1 The treatment involved 16 hours of light followed by 8 hours of darkness. Leaves from seedlings before treatment (day 0) served as a control. Leaves from tissue culture seedlings treated for 3, 5, and 10 days were collected, with three biological replicates per group. Total RNA was extracted. The RNA was then analyzed using Spuddb (…). https: / / spuddb.uga.edu / The website selected 10 potato HSP genes, and the corresponding gene numbers and primers used are shown in Table 1.

[0047] Table 1 Primer sequences for potato HSPs qRT-PCR

[0048]

[0049]

[0050] qRT-PCR analysis of expression levels after high-temperature treatment showed that after 3 days of high-temperature treatment, the expression levels of HSP101, HSP83, HSP70.1, HSP18.2, HSP17.3, and HSP17.7 significantly increased, and after 10 days of treatment, the expression levels of HSP101, HSP83, HSP17.3, and HSP17.7 remained high. Figure 1 A); Further analysis of the heat response elements (HSEs) in the promoter regions of HSP101, HSP83, HSP70.1, and HSP17.3 revealed that the promoter regions of these four genes all contain HSEs, which participate in the heat stress response process. Figure 1 B).

[0051] Further investigation will be conducted on the HSP101 promoter and the HSP70.1 promoter (hereinafter referred to as "HSP70 promoter").

[0052] Example 2

[0053] This embodiment provides the construction of potato heat-induced expression vectors pHSP101:SP6A-GFP, pHSP101:SP6AM-GFP, pHSP70:SP6A-GFP, and pHSP70:SP6AM-GFP, as detailed below:

[0054] Construction method:

[0055] (1) Obtain the promoter sequence of the HSP101 and HSP70 genes in potato from the Spuddb website;

[0056] Using potato E3 leaf DNA as a template, the promoter sequences of HSP101 (primers o25-HSP101P-F and o25-HSP101P-R) and HSP70 (primers o27-HSP70.1P-F and o27-HSP70.1P-R) were amplified by PCR using the ultra-high fidelity DNA polymerase Phanta Max, and the HSP101 promoter fragment and HSP70 promoter fragment were obtained, respectively.

[0057] (2) The potato SP6A gene sequence was obtained from the Spuddb website. Using potato E3 leaf cDNA as a template, the SP6A gene sequence (primers o23-EZR_SP6A-F and o24-EZR_SP6A-R) was amplified by PCR using the high-fidelity DNA polymerase Phanta Max to obtain the potato SP6A gene fragment.

[0058] (3) Based on the binding site of miRNA SES on SP6A, by performing nucleic acid mutation on the primer sequence, the mutant form of potato SP6AM gene fragment with altered nucleic acid sequence but unchanged protein sequence was obtained by two PCRs after SES recognition.

[0059] Specifically, the first PCR: using two primers, o22-SP6AM-R and o23-EZR_SP6A-F, to amplify the intermediate fragment product using potato E3 leaf cDNA as a template;

[0060] Second PCR: Using two primers, o21-SP6AM-F and o24-EZR_SP6A-R, as a template, the intermediate fragment product was used to amplify the mutant form of the potato SP6AM gene fragment that was recognized by SES, with altered nucleic acid sequence but unchanged protein sequence.

[0061] The amplification primers used are shown in Table 2:

[0062] Table 2 Amplification Primer Sequences

[0063]

[0064] The PCR procedure is shown in Table 3:

[0065] Table 3 PCR reaction procedure

[0066]

[0067] (4) The PCR product was obtained and ligated into the enzyme-digested EZR vector (the nucleotide sequence of the EZR vector is shown in SEQ ID NO.34, containing the nucleotide sequence of GFP) by homologous recombination. The enzyme used for recombination ligation was [enzyme name missing]. plus One step PCR Cloning Kit;

[0068] The ligation product pHSP101:SP6A: The EZR vector fragment obtained by digestion with Hind III and Xma I was homologously recombined with the HSP101 promoter fragment obtained in step (1) and the potato SP6A gene fragment obtained in step (2).

[0069] The ligation product pHSP101:SP6AM: The EZR vector fragment obtained by digestion with Hind III and Xma I was homologously recombinated with the HSP101 promoter fragment obtained in step (1) and the potato SP6AM gene fragment obtained in step (3).

[0070] The ligation product pHSP70:SP6A: The EZR vector fragment obtained by digestion with Hind III and Xma I was homologously recombinated with the HSP70 promoter fragment obtained in step (1) and the potato SP6A gene fragment obtained in step (2).

[0071] The ligation product pHSP70:SP6AM: The EZR vector fragment obtained by digestion with Hind III and Xma I was homologously recombinated with the HSP70 promoter fragment obtained in step (1) and the potato SP6AM gene fragment obtained in step (3).

[0072] The ligation products were transformed into DH5α competent cells. Positive strains were selected by resistance (kanamycin) screening and PCR identification, and then sent to a sequencing company for sequencing. Strains with correct sequencing results were retained for future use, yielding expression vectors pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM, respectively. Schematic diagrams are shown below. Figure 2 As shown. The nucleotide sequence of the promoter pHSP101, identified by sequencing, is shown in SEQ ID NO.1; the nucleotide sequence of pHSP70 is shown in SEQ ID NO.2; the nucleotide sequence of SP6A is shown in SEQ ID NO.3, and the protein sequence is shown in SEQ ID NO.4; the nucleotide sequence of SP6AM is shown in SEQ ID NO.5, and the protein sequence is the same as that of SP6A.

[0073] Example 3

[0074] This embodiment provides a method for preparing a potato line with high-temperature tuber formation ability, including the following steps:

[0075] (1) Preparation of bacterial culture: The expression vectors pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM prepared in Example 2 were transformed into Agrobacterium GV3101 by freeze-thaw method to obtain the corresponding engineered bacteria.

[0076] The engineered bacteria were inoculated into 4 mL of YEB liquid medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampin, respectively, and cultured on a shaker at 28 °C and 220 r / min for 24 h to obtain the culture solution;

[0077] Take 3 mL of culture medium into 50 mL of YEB liquid medium containing 50 mg / L Kana and 50 mg / L rifampin, and incubate at 28 °C and 240 r / min on a shaker until the OD600 is 0.5. Then transfer the culture medium to a 50 mL centrifuge tube, centrifuge at 4600 r / min for 6 min, discard the supernatant, and resuspend the precipitate in 50 mL of MS liquid medium containing 3% (w / v) sucrose to obtain Agrobacterium suspension.

[0078] (2) Infection and co-culture: The test tube potatoes that have grown for 10-14 weeks and have a diameter of about 0.5 cm are picked and placed in sterile dishes. They are fixed and cut into 2-3 mm thin slices with flaming forceps and scalpels. All the cut potato slices are transferred with forceps to the Agrobacterium suspension (4 kinds of Agrobacterium suspension) obtained in step (1) and soaked for 10 min. Then the bacterial suspension is discarded, and the potato slices are transferred to a culture dish with sterile filter paper to absorb the bacterial liquid on the surface of the potato slices. After 20 min, they are transferred to the co-culture medium and placed in a dark incubator at 25℃ for 2 days.

[0079] The co-culture medium was based on MS basal medium, supplemented with 3% sucrose (w / v), 0.2 mg / L IAA, 0.2 mg / L GA3, 0.5 mg / L 6-BA, and 2 mg / L ZT (all final concentrations).

[0080] (3) Bud differentiation and rooting: The potato slices cultured in step (2) were transferred to a co-culture medium containing 5 mg / L hygromycin, 200 mg / L cephalosporin and 200 mg / L termethin, and cultured under conditions of 2000 lx light intensity, 16 h light + 8 h dark photoperiod, and 23 ± 1 ℃ temperature. After 21 to 28 days, resistant buds grew from the center of the test tube potato slices. When the resistant buds reached 1.5 cm to 2 cm in length, they were cut off and transferred to MS liquid medium containing 5 mg / L hygromycin and 200 mg / L cephalosporin for rooting and selection culture, thereby obtaining transgenic potato seedlings (pHSP101:SP6A potato transgenic seedlings, pHSP101:SP6AM potato transgenic seedlings, pHSP70:SP6A potato transgenic seedlings, and pHSP70:SP6AM potato transgenic seedlings, respectively).

[0081] (4) Identification of positive plants: Genomic DNA was extracted from four types of transgenic potato seedlings using the CTAB method, and PCR was performed to identify the exogenous inserted fragments. The primers used for identification are shown in Table 4.

[0082] Table 4 Primers for identifying transgenic seedlings

[0083]

[0084] Among them, the pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM potato transgenic seedlings were successfully identified and will be used for further research.

[0085] Example 4

[0086] This embodiment provides a laser confocal microscopy method for identifying the expression level of GFP protein in thermally induced expression lines.

[0087] The potato transgenic seedlings (pHSP101:SP6A potato transgenic seedlings, pHSP101:SP6AM potato transgenic seedlings, pHSP70:SP6A potato transgenic seedlings, pHSP70:SP6AM potato transgenic seedlings) successfully identified in Example 3 and the E3 wild-type seedlings tissue culture seedlings grown for 3 weeks were subjected to high sugar induction to form tubers.

[0088] Specifically, robust seedlings of pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM potato transgenic seedlings were selected. These seedlings were then cut into stem segments and inserted into MS liquid medium containing 8% (w / v) sucrose and 2‰ (w / v) activated carbon. The culture conditions were a light intensity of 83 μmol / m². - 2 s -1 After 8 hours of light exposure followed by 16 hours of darkness (photocycle), potato tubers induced for 6 weeks were examined under a laser confocal microscope to observe GFP fluorescence. The GFP fusion protein expression results are as follows: Figure 3 As shown, this indicates that GFP protein was successfully expressed in all four types of transgenic potato seedlings.

[0089] Example 5

[0090] This embodiment provides phenotypic identification of heat-induced expression lines after in vitro tuber formation under high-temperature treatment.

[0091] High-sugar induction and high-temperature treatment experiments were conducted on the successfully identified potato transgenic seedlings (pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM) and E3 wild-type seedlings that had grown for 3 weeks. Strong seedlings from the pHSP101:SP6A, pHSP101:SP6AM, pHSP70:SP6A, and pHSP70:SP6AM seedlings were selected, and stem segments were inserted into MS liquid medium containing 8% (w / v) sucrose and 2‰ (w / v) activated carbon. The culture conditions were a light intensity of 83 μmol / m². -2 s -1 The culture was conducted with 8 hours of light followed by 16 hours of darkness (photocycle). Two temperature groups were established: room temperature (22℃ light + 18℃ darkness) and high temperature (30℃ light + 28℃ darkness). Each line was placed in 2-3 boxes, with 5-9 plants per box. After 8 weeks of continuous high-temperature treatment, the tuber formation phenotype of the tissue culture seedlings was observed and statistically analyzed. The plant phenotypes are as follows: Figure 4 As shown in Tables 5 and 6, the tuber formation rate statistics of the expression lines are as follows: pHSP101:SP6A-1 (labeled as 101A-1), pHSP101:SP6AM-11 (labeled as 101M-11), and pHSP70:SP6AM-8 (labeled as 70M-8) all have the ability to form tubers at high temperatures after in vitro induction of tubers and high temperature treatment.

[0092] Table 5. Statistics on tuber formation in vitro at room temperature for transgenic lines.

[0093]

[0094] Table 6. Statistics on tuber formation in vitro after high-temperature treatment of transgenic lines.

[0095]

[0096]

[0097] Example 6

[0098] This embodiment provides identification of tuber formation phenotype in heat-induced expression lines.

[0099] Plantlets of the heat-resistant lines (pHSP101:SP6A-1, pHSP101:SP6AM-11, and pHSP70:SP6AM-8) initially identified through high-sugar-induced tuber formation were transplanted into nutrient soil after 3 weeks of cultivation. Tuber formation was induced at 22℃ with 16 hours of light and 8 hours of darkness. After 4 weeks of growth, tuber formation was induced again with 8 hours of light and 16 hours of darkness. Three plants were treated at room temperature (22℃ light / 18℃ darkness), and three plants were treated at high temperature (31℃ light / 28℃ darkness). Tuber phenotypes were analyzed at 12 weeks. Tuber phenotypes are as follows: Figure 5 As shown, the results indicate that, compared with wild-type E3, the pHSP101:SP6A-1, pHSP101:SP6AM-11, and pHSP70:SP6AM-8 lines have stronger tuber-forming ability under high temperature.

[0100] Example 7

[0101] This embodiment provides identification of tuber formation phenotype and gene expression analysis of heat-induced expression lines in field trials during high-temperature weather.

[0102] The pHSP101:SP6A-1, pHSP101:SP6AM-11, pHSP70:SP6A-14, and pHSP70:SP6AM-8 lines were transplanted into a greenhouse from April 12, 2024 to August 14, 2024 (Shanghai). During the seedling stage (early May), the daytime temperature inside the greenhouse reached 30℃. After full tuber formation, the tuber phenotype of the expression lines under high temperature was tested. The tuber phenotype and statistical results are as follows: Figure 6 As shown, the relative expression level analysis of the SP6A gene is as follows: Figure 7 As shown in the figure. Field trials showed that, compared with the wild type, the pHSP101:SP6A-1, pHSP101:SP6AM-11, pHSP70:SP6A-14, and pHSP70:SP6AM-8 lines exhibited significantly increased tuber number per plant, maximum tuber weight, and tuber starch content under high-temperature conditions. SP6A gene expression was also significantly induced. Therefore, the pHSP101:SP6A-1, pHSP101:SP6AM-11, pHSP70:SP6A-14, and pHSP70:SP6AM-8 lines possess strong heat-resistant tuber formation capabilities and also increase tuber starch content. These lines can be used for the breeding of heat-resistant potato varieties, laying the foundation for further research on the tuber development mechanism of potatoes under high temperatures.

[0103] Specifically, SEQ ID NO.1 (5'-3') is shown below:

[0104]

[0105] SEQ ID NO.2 (5'-3') is shown below:

[0106]

[0107] SEQ ID NO. 3 (5’-3’) is as follows:

[0108] atgcctagagttgatccattgatagttggtcgtgtgataggtgatgttttagatccattcactaggtctgttgatcttagagttgtttataataataaagatgtgaacaatgcatgtgtgttgaaaccttcacaagttgttatgcaacctagggttcatattggaggggacgatcttcgcaacttttacactctgattatggtggatcctgatgctccaagcccaagcgaccctaacttgagggagtatctacattggctggtcacagatatcccagcaactacaaatacaagctttggaaatgaagtcgtatgctacgagaatccaacacctacgatgggaattcatcgattcgttttggttttatttcgacaatcaagacgtgaaacagtgtatgccccaggttggcgtcaaaatttcaacacaagagactttgctgagctttacaatcttggattgcctgttgcagctgtttacttcaattcccatagggagagtggcactggaggacgtcgcgca

[0109] SEQ ID NO. 4 is as follows:

[0110] MPRVDPLIVGRVIGDVLDPFTRSVDLRVVYNNKDVNNACVLKPSQVVMQPRVHIGGDDLRNFYTLIMVDPDAPSPSDPNLREYLHWLVTDIPATTNTSFGNEVVCYENPTPTMGIHRFVLVLFRQSRRETVYAPGWRQNFNTRDFAELYNLGLPVAAVYFNSHRESGTGGRRA

[0111] SEQ ID NO. 5 (5’-3’) is as follows:

[0112] atgcctagagttgatccattgatagttggtcgtgtgataggtgatgttttagatccattcactaggtctgttgatcttagagttgtttataataataaagatgtgaacaatgcttgcgtgcttaagccatcacaagttgttatgcaacctagggttcatattggaggggacgatcttcgcaacttttacactctgattatggtggatcctgatgctccaagcccaagcgaccctaacttgagggagtatctacattggctggtcacagatatcccagcaactacaaatacaagctttggaaatgaagtcgtatgctacgagaatccaacacctacgatgggaattcatcgattcgttttggttttatttcgacaatcaagacgtgaaacagtgtatgccccaggttggcgtcaaaatttcaacacaagagactttgctgagctttacaatcttggattgcctgttgcagctgtttacttcaattcccatagggagagtggcactggaggacgtcgcgca

[0113] SEQ ID NO.34 (5'-3') is as follows:

[0114]

[0115] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. Use of a high temperature inducible promoter in the regulation of potato breeding, characterized in that, The high-temperature inducible promoter is selected from either pHSP101 or pHSP70; The nucleotide sequence of pHSP101 is shown in SEQ ID NO.1; The nucleotide sequence of pHSP70 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Application of the high-temperature inducible promoter in promoting tuber formation in potatoes at both room temperature and high temperature and / or increasing the starch content of potato tubers.

3. The application of a heat-inducible expression vector in potato breeding regulation, characterized in that, The heat-inducible expression vector includes the high-temperature inducible promoter and potato tuber gene as described in claim 1; The potato tuber gene is selected from either the SP6A gene or the SP6AM gene. The nucleotide sequence of the SP6A gene is shown in SEQ ID NO.3; The nucleotide sequence of the SP6AM gene is shown in SEQ ID NO.

5.

4. The application according to claim 3, characterized in that, Application of the heat-inducible expression vector in promoting tuber formation in potatoes at both room temperature and high temperature and / or increasing the starch content of potato tubers.

5. An application of heat-induced host cells in potato breeding regulation, characterized in that, The heat-induced host cell includes the heat-induced expression vector as described in claim 3.

6. The application according to claim 5, characterized in that, The application of heat-induced host cells in promoting tuber formation in potatoes at both room temperature and high temperature and / or increasing the starch content of potato tubers.

7. A method for preparing a potato strain with high-temperature tuber formation ability, characterized in that, Includes the following steps: (S1) The high-temperature inducible promoter and the potato tuber gene were inserted into the basic vector to construct a recombinant vector containing the high-temperature inducible promoter and the potato tuber gene; (S2) A recombinant vector containing a high-temperature inducible promoter and a potato tuber gene was transferred into Agrobacterium and then infected with potato micro-tuber chips to induce bud differentiation, thus obtaining a potato line with the ability to form tubers at high temperatures. The high-temperature inducible promoter is selected from either pHSP101 or pHSP70. The nucleotide sequence of pHSP101 is shown in SEQ ID NO.1; The nucleotide sequence of pHSP70 is shown in SEQ ID NO.

2.

8. The method for preparing a potato line with high-temperature tuber formation ability according to claim 7, characterized in that, In step (S1), the potato tuber gene is selected from either the SP6A gene or the SP6AM gene.

9. The method for preparing a potato strain with high-temperature tuber formation ability according to claim 8, characterized in that, The nucleotide sequence of the SP6A gene is shown in SEQ ID NO.3; The nucleotide sequence of the SP6AM gene is shown in SEQ ID NO.

5.

10. A potato strain with the ability to form tubers at high temperatures, characterized in that, It is prepared by any one of the methods described in claims 7 to 9.

Images